Drug-Resistant Hematological Malignancies Organoids Can be Established By Magic-PDX Model Strategy

Background:

Organoids are crucial for tumor mechanism study and new drug development. Studies showed that Patient-derived xenograft (PDX) models are helpful for the subsequent construction of organoids. However, the fabrication of either PDXs and organoids remain challenging for hematological malignancies (HM). Traditional PDX fabrication through tail vein injection (IV-PDX) succeeds with probability 10%-40%, and needs 4-6 months. In order to solve this problem, we developed a new model of PDX, helping magnetically-induced-cells (MagIC) home to bone marrow (BM), which is named as MagIC-PDX. It shows clinical features of HM and only needs weeks to fabricate, after which we can easily harvest BM with expanded malignant cells.

Objective:

To explore the best experimental elements combination for constructing organoids of HM with the BM of MagIC-PDX Strategy.

Methods:

1.1 Primary BM isolation and magnetization: Isolate the BM from drug-resistant HM (acute leukemia or multiple myeloma etc.), mononuclear cells were separated by Ficoll and labelled overnight with recombinant lentiviral vectors carrying luciferase and GFP, then the cells were non-specifically magnetized with the “magnetic nanomotors”for 30mins, at 37 ℃.

1.2 PDX modeling: the magnetized cells were inoculatedinto the right femur of 6-8 w NSG female mice by our patented needle (CN205460216U) under a precise magnetic field overnight to help the target homing of malignant cells to BM. The survival, peripheral blood count and weight of mice were monitored, and tumor load and metastasis were studied with bioluminescence at least once a week. BM puncture were performed weekly with the same patented needle, then studied by BM smears, flow cytometry, FISH and qPCR (MICM), the MICM information, and compared with the clinical data.

1.3 T-Trap treatment: CD4/TGF-βbi-specific antibody (T-Trap, Majory, China) were applied 300 µg p. to the mice 2 weeks before the BM harvesting, untreated mice were set as control group.

1.4 Organoids Construction: Extract BM on d28 and culture the cells with “magnetic nanorobots” for 30mins at 37℃, then the magnetized BM were cultured in the AMO Organoids culture system for the organoids with or without precise maegnetic fields, and the number of oganoids per million cells with a diameter more than 2.5mm (d>2.5mm) on d4 were recorded.

1.5 Grouping and data statasis: Four groups of experiments were compared, includes T-Trap, MagIC, T-Trap+MagIC and negative control group.

Results:

2.1 The magnetization rate of BM cells are (87.8±8.1)%, n=5. The MagIC-PDX model can be fabricated within one week, with success rates (95.3±6.5)%, n=5, showing consistent and stable bioluminescent signals at the injection site, which expanded over time. The traditional IV-PDX requires at least 3 weeks until the detection of unevenly distributed signals, and has a much shorter lifespan (28.4±2.7 vs 62.4±4.8)d, leading to a much smaller experimental window (14.4±2.7 vs 59.4±4.8)d than MagIC-PDX group.

2.2 Organoids formation: The combination of T-Trap+MagIC in the AMO Organoids culture system performed the best: Every million cells, the number of d>2.5mm organoids on d4 among the four different groups (T-Trap+MagIC, MagIC, T-Trap, and negative control group (n=5)) are 22.6±16.4, 6.0±1.6, 2.0±1.9 and 0±0 respectively. The T-Trap+MagIC and MagIC group had significantly better results than pure T-Trap or negative groups (p<0.01).

Discussion:

The acceleration of PDX modeling and organoid construction from HM by T-Trap+MagIC technology could be attributed to the following mechanisms: a) The non-specific homing of HM cells in mouse bone marrow, b) rapid expansion of malignant cells facilitating organoid construction, and c) the increase of local collagen in BM by T-Trap, and the magnetic force integrating the cells by MagIC.

Conclusion:

MagIC-PDX Model combined with T-Trap is a new organoid-forming method of drug-resistant HM, which will facilitate the future new drugs' development, mechanisms study, and personalized medicine for malignancies.

No relevant conflicts of interest to declare.